Effect of DHA treatment on PIEZO1-GFP. The fatty acid DHA is an important dietary polyunsaturated fatty acid that is thought to enhance separation of lipid phases in vitro and confer fluidity to disordered lipid phases (Levental et al., 2016). The fatty acid becomes incorporated into membrane phospholipids when supplied long term in the cell culture medium (Shaikh et al., 2009). (A) Patch-clamp recording of PIEZO1-GFP in cell-attached mode, 24-h treatment with 25 µM DHA recorded at +65 mV pipette voltage. Each current plot and its respective pressure trace are color-coded. (B) Boltzmann fit of normalized current versus pressure plot of PIEZO1-GFP after 24- and 48-h treatment with 25 µM DHA. Control (n = 8 cells), DHA 24 h (n = 5 cells), DHA 48 h (n = 6 cells). (C) Quantification of P50 values derived from individual Bolzmann functions analyzed in B. (D) Average peak current latency time recorded in the −20 to −70 mmHg pressure regimes for control and DHA-treated cells. Control (n = 8 cells), DHA 24 h (n = 5 cells), DHA 48 h (n = 6 cells). (E) Quantification of PIEZO1-GFP current inactivation represented as normalized (Norm.) steady-state current at all pressures between −20 and −70 mmHg applied pressure in control, 24- and 48-h DHA-treated cells. Control PIEZO1-GFP (n = 8 cells), DHA 24 h (n = 5 cells), DHA 48 h (n = 3 cells). Statistical analysis was performed using one-way ANOVA; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; error bars represent SEM.
